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dc.contributorYau-Heiu Hsuen_US
dc.contributorHau - Ren Chenen_US
dc.contributor.advisorMeng hsiao Mengen_US
dc.contributor.authorRen, Jia-xianen_US
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dc.description.abstract竹嵌紋病毒 (Bamboo mosaic virus, BaMV) 為單股正意RNA病毒,基因體含有五個轉譯架構 (open reading frames, ORFs),ORF1可轉譯出複製酵素,其餘ORFs轉譯出非複製酵素的蛋白質。ORF2、ORF3及ORF4為三個互相重疊的基因組成,分別轉譯出的蛋白質 (triple gene block proteins, TGBp1、TGBp2、TGBp3) 與病毒在寄主細胞間之移動有關,ORF5轉譯出病毒包被所需的外鞘蛋白質 (coat protein, CP)。關於這些非複製酵素的蛋白質是否與寄主蛋白質參與交互作用,目前尚不清楚。此外,本實驗室已證實菸草中的一個甲基轉移酶 (methyltransferase in Nicotiana benthamiana, NbMTS1) 與病毒的RNA聚合酵素 (RNA-dependent RNA polymerase, RdRp) 具有交互作用,可抑制病毒感染的能力,由於不排除NbMTS1是透過影響細胞之生理機能而導致病毒複製量下降的原因,因此尋找與NbMTS1有交互作用的寄主蛋白質,有助於釐清NbMTS1抑制病毒複製的詳細機制。本研究為尋找在病毒感染過程中可能與TGBp及CP產生交互作用的寄主蛋白質,以及尋找與NbMTS1具交互作用的蛋白質,因此以TGBp、CP及NbMTS1作為釣餌蛋白質 (bait protein),利用酵母菌雙雜交篩選法 (yeast two-hybrid screen),從菸草葉子的cDNA基因庫中尋找能與釣餌蛋白質交互作用的蛋白質,為了確保完整篩選到所有的cDNA基因庫,每個釣餌蛋白質至少分別進行三重複以上大規模酵母菌轉型作用來篩選cDNA基因庫。TGBp3無法在酵母菌中表現融合蛋白質,因此無法作為此系統的釣餌蛋白質。以TGBp1、TGBp2及NbMTS1作為釣餌蛋白質來篩選菸草葉子cDNA基因庫時,篩選出的質體於再次確認後皆為偽陽性。另一方面,以CP作為釣餌蛋白質時,將篩選到生長在缺乏組胺酸培養基中的菌落,利用β-galactosidase活性測試,篩選出活性較強之轉殖株;經由再次確認質體所轉譯出的蛋白質與CP具有專一性的交互作用。將這些質體定序之後,與蛋白質資料庫比對,發現篩選到的蛋白質為病毒本身的CP,且N端已去除30個胺基酸,顯示竹嵌紋病毒之缺乏N端30個胺基酸CP,不會影響CP間的交互作用。zh_TW
dc.description.abstractBamboo mosaic virus (BaMV), a plus-sense RNA virus, has a genome that consists of five open reading frames (ORFs). ORF1 encodes the viral replicase proteins. ORF2, ORF3, and ORF4 encode triple gene block proteins respectively, (TGBp1, TGBp2, and TGBp3) which are involved in viral movement. ORF5 encodes the viral coat protein (CP). To date, the interaction between the non-replicase proteins and host proteins is not clear yet. The previous study had evidenced the interaction between a putative methyltransferase (NbMTS1) of Nicotiana benthamiana and RNA-dependent RNA polymerase domain (RdRp domain), and the NbMTS1 can inhibit the viral infection, suggesting that it may be a part of the defense mechanism in plant. Since that NbMTS1 can decrease the viral replication, we wondered whether the inhibitory effect is mediated through interaction with other cellular protein. To investigate the host proteins which may interact with CP or TGBp, the TGBp, CP, and NbMTS1 was used as bait proteins in yeast two-hybrid screen to find out the interactive proteins from a leaf cDNA library of N. benthamiana. Each bait protein was executed large-scale library transformation for three times respectively to confirm that we can select all of cDNA library. At first, we used TGBp3 as the bait protein, but it could not be expressed in the yeast. Then we used TGBp1, TGBp2, and NbMTS1. The result of the selected plasmids was all false positive. Finally, we chose CP to be the bait protein. We used some colonies which grew in the absence of histidine medium, and then tested them by β-galactosidase activity assay to select out some stronger interaction. After double checked, we transformed the plasmids to the yeast so that the proteins could interact with CP specifically. We did a DNA sequencing comparison in NCBI BLAST and found that thee proteins were truncated CP; moreover, their N-terminal had already deleted 30 amino acids. This shows the CP-CP interaction would not be affected when the viral CP lacked of N-terminal 30 amino acids.en_US
dc.description.tableofcontents目次 中文摘要 i 英文摘要 ii 第一章、前言 1 竹嵌紋病毒 1 找尋寄主因子 1 植物病毒的移動蛋白質 2 植物病毒的外鞘蛋白質 3 菸草中的甲基轉移酶 3 酵母菌雙雜交系統 4 研究目的 4 第二章、材料與方法 5 第一節、菌株、載體與培養基 5 第二節、質體之構築 5 第三節、抽取大腸桿菌之質體DNA 7 第四節、大腸桿菌Top10F’之轉型作用 (transformation) 7 第五節、酵母菌L40之轉型作用 (small-scale yeast transformation) 8 第六節、抽取酵母菌之質體DNA 8 第七節、萃取酵母菌之蛋白質 9 第八節、西方墨點法 (Western Blot) 9 第九節、大量抽取大腸桿菌之質體DNA 10 第十節、大規模酵母菌之轉型作用 (large-scale library transformation) 11 第十一節、β-galactosidase定性分析 (colony-filter assay) 12 第三章、結果 13 第一節、釣餌質體之構築、融合蛋白質的表現與非專一性活化的測試 13 一、釣餌質體pHybLex-TGBp/Zeo與pHybLex-CP/Zeo之構築 13 二、以酵母菌表現LexA-TGBp與LexA-CP融合蛋白質 13 三、釣餌蛋白質TGBp與CP之非專一性活化的測試 13 四、釣餌質體pHybLex-NbMTS1(ΔN36)/Zeo與pHybLex-NbMTS1(ΔN105)/Zeo之構築 14 五、以酵母菌表現LexA-NbMTS1(ΔN36) 與LexA-NbMTS1(ΔN105) 融合蛋白質 14 六、釣餌蛋白質NbMTS1(ΔN36) 與NbMTS1(ΔN105) 之非專一性的活化測試 15 第二節、cDNA基因庫的來源 15 第三節、酵母菌雙雜交篩選法之執行 15 一、製備大量cDNA基因庫質體 15 二、以酵母菌雙雜交法於菸草葉子cDNA基因庫中篩選與TGBp1及TGBp2交互作用之蛋白質 15 三、以酵母菌雙雜交法於菸草葉子cDNA基因庫中篩選與CP交互作用之蛋白質 16 四、以酵母菌雙雜交法於菸草葉子cDNA基因庫中篩選與NbMTS1(ΔN36) 交互作用之蛋白質 17 第四章、討論 19 參考文獻 23 表目次 表1、PCR所用引子之序列 29 表2、以TGBp1為釣餌蛋白質執行酵母菌雙雜交法篩選之結果 30 表3、以TGBp2為釣餌蛋白質執行酵母菌雙雜交法篩選之結果 31 表4、以CP為釣餌蛋白質執行酵母菌雙雜交法篩選之結果 32 表5、以NbMTS1(ΔN36) 為釣餌蛋白質執行酵母菌雙雜交法篩選之結果 33 圖目次 圖1、將LexA-TGBp及LexA-CP於酵母菌中表現融合蛋白質 34 圖2、釣餌蛋白質TGBp與CP非專一性活化報導基因的測試 35 圖3、NbMTS1的不同截斷蛋白質 36 圖4、將LexA-NbMTS1(ΔN36) 及LexA-NbMTS1(ΔN105) 於酵母菌中表現融合蛋白質 37 圖5、釣餌蛋白質NbMTS1(ΔN36) 與NbMTS1(ΔN105) 非專一性活化報導基因的測試 38 圖6、測試以CP為釣餌蛋白質進行酵母菌雙雜交法篩選到之酵母菌轉型株的HIS3與LacZ兩種報導基因的表現 39 圖7、測試NbMTS1(ΔN36) 釣餌蛋白質與RdRp是否具有交互作用 40 附錄目次 附錄1、竹嵌紋病毒基因體及其所轉譯出之蛋白質 41 附錄2、菸草甲基轉移酶的示意圖 42 附錄3、酵母菌雙雜交系統之模式圖 43 附錄4、pHybLex/Zeo質體之圖譜與特徵 44 附錄5、pYESTrp2質體之圖譜及特徵 45 附錄6、細菌低鹽培養基LSLB 46 附錄7、酵母菌完全培養基YPAD 46 附錄8、酵母菌營養篩選培養基YC 47zh_TW
dc.subjectBamboo mosaic virusen_US
dc.subjectYeast Two-hybrid Screenen_US
dc.title以竹嵌紋病毒非複製酵素的蛋白質及菸草甲基轉移酶 (NbMTS1) 作為釣餌蛋白質利用酵母菌雙雜交法尋找菸草中具交互作用之蛋白質zh_TW
dc.titleSearching the Proteins Interacted with the Non-replicase Proteins of Bamboo Mosaic Virus and the Methyltransferase of Nicotiana benthamiana (NbMTS1) by Yeast Two-hybrid Screenen_US
dc.typeThesis and Dissertationzh_TW
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